From ancient times, mushrooms of basidiomycetes, such as Lentinus edodes, Trichloma matsutake and Flammulina velutipes, have been used for foods, and some of them, e.g., Polyporaceae of basidiomycetes, have been valuably used as Chinese medicines.
Meanwhile, various methods to extract active ingredients from the basidiomycetes have been proposed. For example, Japanese Patent Laid-Open Publication No. 46859/1979 discloses a method for preparing a healthful food comprising the steps of inoculating basidiomycetes in a culture medium mainly composed of bagasse, proliferating the mycelium and squeezing the culture medium containing proliferated mycelium to obtain active ingredients.
The effect of the bagasse of the culture medium used in this method is described in, for example, a monograph by Sutemi Oka, et al., "ANTITUMOR ACTIVITY OF SOME PLANT POLYSACCHARIDES (FRACTIONATION AND ANTITUMOR ACTIVITY OF BAGASSE POLYSACCHARIDE)", GANN, Vol. 59, pp. 35 to 42, February, 1968. In more detail, it is described that a fraction obtained from a crude powder of bagasse polysaccharide contains galactose, arabinose, xylose and mannose along with a small amount of glucose and intraperitoneal injections of the fraction resulted in remarkable inhibitory effect on the growth of subcutaneously implanted Sarcoma 180 (kind of sarcoma, malignant tumor) in mice. Therefore, health effects given by intake of ingredients of the bagasse can be expected.
In the method described in the above publication, however, there is a problem in that the extraction efficiency is bad because concentrations of the active ingredients in the separate liquid are low and hence a complicated concentrating operation is necessary.
The present inventors have studied to solve such a problem as mentioned above and proposed, in Japanese Patent Application No. 34123/1987, "a method for extracting useful ingredients from Flammulina velutipes mycelium and a bagasse culture medium, comprising the steps of inoculating Flammulina velutipes fungus in a solid culture medium containing bagasse as a substrate, then disentangling the solid culture medium containing proliferated mycelium in such a manner that the amount of the 12-mesh passing disentangled culture medium becomes not more than 30 w/w %, adding water and at least one enzyme selected from the group consisting of cellulase, protease and glucosidase to the disentangled solid culture medium, crushing and grinding the solid culture medium in the presence of the enzyme so that the amount of the 12-mesh passing bagasse fibers becomes at least 70 w/w %, then heating up to a temperature of 95.degree. C. to inactivate the enzyme and perform sterilization".
The present inventors have further proposed, in Japanese Patent Publication No. 23826/1985, a method for preparing a healthful drink comprising the same steps as in the method of Japanese Patent Application No. 34123/1987 except that Lentinus edodes fungus is used as the fungus to be inoculated.
The present inventors have furthermore proposed a method for preparing a healthful drink using Ganoderuma lucidium fungus in Japanese Patent Application No. 5355/1984 (Japanese Patent Publication No. 35149/1992) and Japanese Patent Application No. 5356/1984 (Japanese Patent Publication No. 6171/1992).
According to the methods proposed by the present inventors, useful ingredients can be extracted in high concentrations from the mycelia (Flammulina velutipes mycelium, Lentinus edodes mycelium and Ganoderuma lucidium mycelium) and the bagasse culture media.
In these methods, however, repeated heat treatments are generally carried out from the viewpoint of extraction efficiency. For example, the crushing-grinding operation of the solid culture medium having been added with an enzyme is carried out under heating at a temperature of usually 30 to 50.degree. C., the inactivation of enzyme is carried out under heating at a temperature of up to 95.degree. C., and also the sterilization is carried out under heating.
The present inventors have earnestly studied to further improve the extraction efficiency, and they have found that useful ingredients such as .beta.-glucan can be efficiently extracted from basidiomycetous mycelium and a bagasse solid culture medium by:
a method comprising the steps of squeezing a bagasse culture medium containing proliferated basidiomycetous mycelium to separate it into a solid component containing mycelium and a squeeze liquid and subjecting the mycelium-containing solid component to a specific treatment, or PA1 a method comprising the steps of disentangling a bagasse culture medium containing proliferated basidiomycetous mycelium, contacting the mycelium-containing bagasse culture medium with a specific enzyme (i.e., allowing a specific enzyme to act on the culture medium) under specific conditions and heating the resulting cell wall lysis product-containing liquid to inactivate the enzyme and perform sterilization. PA1 inoculating basidiomycetes in a solid culture medium containing bagasse as a substrate and proliferating mycelium, and then squeezing the solid culture medium containing proliferated mycelium to obtain a squeeze liquid (i), PA1 separately, adding water and an enzyme (preferably enzymes containing a cell wall lytic enzyme) to a mycelium-containing solid residue (solid component) having been separated from the squeeze liquid (i), then stirring the resulting mixture while maintaining the mixture at 30 to 60.degree. C. to lyse (decompose) mycelial cell walls, and PA1 heating the resulting cell wall lysis product-containing liquid (ii) up to a temperature of 95.degree. C. to inactivate the enzyme and perform sterilization. PA1 inoculating basidiomycetes in a solid culture medium containing bagasse as a substrate and proliferating mycelium, and then squeezing the solid culture medium containing proliferated mycelium to obtain a squeeze liquid (i), PA1 separately, disentangling a mycelium-containing solid residue (solid component) having been separated from the squeeze liquid (i), PA1 adding water and an enzyme (preferably enzymes containing a cell wall lytic enzyme) to the disentangled solid residue (solid component), then crushing and grinding the solid component while maintaining the mixture at 30 to 60.degree. C. to lyse mycelial cell walls, and PA1 heating the resulting cell wall lysis product-containing liquid (ii) up to a temperature of 95.degree. C. to inactivate the enzyme and perform sterilization. PA1 inoculating basidiomycetes in a solid culture medium containing bagasse as a substrate and proliferating mycelium, and then disentangling the solid culture medium containing proliferated mycelium, PA1 contacting the disentangled solid culture medium with a .beta.-1,3-glucanase-containing liquid (a) at a temperature of 30 to 60.degree. C. for 1 to 65 minutes to lyse mycelial cell walls, and PA1 heating the resulting cell wall lysis product-containing liquid up to a temperature of 95.degree. C. to inactivate the enzyme and perform sterilization. PA1 mushrooms of Agaricales, which are generally used for foods, such as Trichloma matsutake, Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus, Pholiota nameko, Boletus, Lyophyllum and Lactarius volemus; PA1 mushrooms of Polyporales, which are used as Japanese and Chinese medicines or used for foods, such as Fomes applanatus, Fomes piniocola, Coriolus versicolor, Ganoderuma lucidium and Grifola frondosa; and PA1 mushrooms of Auriculariales and Tremellales, which are generally used for foods, such as Auricularia and Tremella.
Based on the finding, the present invention has been accomplished.
In a monograph entitled "On The New Cell Wall Lytic Enzymes" (by Shinichiro Shimada, New Food Industry, Vol. 28, No. 8 (1986)), it is described that mycelium obtained by complete culturing of each mushroom of Pleurotus ostreatus and Flammulina velutipes is treated with a 2% solution (pH: 5.6) of Fanselase (trade name of enzyme reagent, available from Yakult K.K.) under the temperature conditions of 30.degree. C. for 1 or 2 hours to thereby produce protoplast. It is further described that protoplast is produced also from Lentinus edodes mycelium.
In the above monograph, however, there is not so much as suggestion on the methods to efficiently extract the useful ingredients such as .beta.-glucan from the basidiomycetous mycelia and the bagasse culture media. Actually, even if the above mycelia are contacted with Fanselase under the above temperature conditions for a long period of time, the useful ingredients such as .beta.-glucan having antitumor activity cannot be efficiently extracted from the basidiomycetous mycelia and the bagasse culture media.